臨床上需要對長段尿道及輸尿管缺損患者進行組織重建。然而目前並無個人化平台以及適合之生物相容性材質可作為組織重建之用。期許高生物相容性支架能做為臨床泌尿道創傷患者組織再生可用之材料。靜電紡絲系統 (Electrospinning)為近年新興的奈米纖維製程技術，可直接且快速的將材料紡絲成奈米纖維，提高比表面積、孔隙率及具備有微奈米級孔洞結構，可根據材料的多元化應用在不同的領域；三維列印技術則能提供品質均一甚至客製化的支架。我們選用具有良好生物相容性之聚己內酯 (Polycaprolactone, PCL)及蠶絲蛋白 (SF)作為生物支架的材料，比較靜電紡絲 (ES)及三維列印結合靜電紡絲(3D-ES)為平台所製生物支架其物化性及生物相容性之差異。本實驗從蠶繭中萃取出蠶絲蛋白後再使用靜電紡絲系統及三維列印技術印製奈米纖維支架後，除了觀察材料表面結構以及物理化學性測試，另針對細胞的生物相容性及穿透能力作討論。在體外組織模式中，首先觀察細胞生長貼附於支架上情況，以及活體組織培養之狀況。在紐西蘭大白兔的動物模式中，進行單側輸尿管進行1/5置換(兔子輸尿管全長大約10-12公分)，經6、7週後犧牲動物進行組織切片，並利用HE 及 Masson染色觀察其貼附生長情況。結果發現，在物理特性測試中，當SF的比例增加時，PCL-SF支架中奈米纖維孔的直徑和尺寸均增加、拉伸強度下降，降解速度無太大差異。在傅里葉轉換紅外光譜 (FTIR)分析中可以觀察到PCL及SF的對應譜峰，MTT結果表明，SF在支架中的比例增加有利於細胞增殖，總和上述條件我們選出PCL:SF=4：6為最佳混和比例。由於奈米纖維的直徑增加及孔洞的大小增加，觀察到結合三維列印後細胞在支架上的增殖速度明顯提升。離體組織實驗中PCL-SF支架顯示細胞可沿支架穩定生長兩週，且細胞大多沿著外緣生長。動物模式方面，在經過6、7週後可觀察到不同細胞沿著管壁外緣生長，由內而外依序為黏膜層、黏膜下層、肌肉層與漿膜層，而管壁內緣在近腎端只有黏膜層貼附生長，在膀胱端黏膜及肌肉層都有生長，但是在生物支架的內緣中段在7週的時間中細胞尚未生長貼附。
本實驗證明 利用三維列印結合靜電紡絲系統可製作出適合細胞組織生長之4:6 PCL-SF支架，並在動物實驗中達到輸尿管重建之目的， 因此此類新載體可做為將來臨床泌尿道系統組織重建之應用。

Clinically, tissue reconstruction is needed in patients with urethral and ureteral injury with long segment defects. However, there is no customized platform and suitable bio-compatibility materials to produce the tissue regeneration vehicle currently. We hope this customized scaffold could be a useful replacement material in tissue regeneration for urological trauma patients. Electrospinning (ES) system is a new nanofiber process technology in recent years. It can directly and quickly spin the material into nanofibers, improve the specific surface area, porosity and micro/nano-scale pore structure. 3D printing technology can provide uniform and customized brackets in different fields. Our study chooses highly bio-compatibility materials, silk fibroin (SF) and polycaprolactone (PCL) as bio-materials scaffolds respectively. The differences in physicochemical properties and biocompatibility of bio-scaffolds prepared by ES and 3D printing combined with electrospinning (3D-ES) were compared.
SF powder was prepared from dissolved cocoons, then extracted by dialysis and lyophilized before study. Various ratios of PCL and SF nanofiber in formic acid were generated by ES or self-designed 3D-ES platform. The physical and biological characteristics were assayed by SEM, degradation test, tension test and cell attachment assay of fibroblasts and urothelial cells on various nanofibers of PCL-SF. The ex vivo resected ureteral tissue was anastomosed with the PCL-SF scaffolds and cultured in ex vivo bath for two weeks. The cellular growth on scaffold was observed microscopically by HE stain. In the New Zealand white rabbit model, we performed a 1/5 ratio (2 cm) replacement of the unilateral ureter(rabbit ureter is about 10 cm length). After 6 and 7 weeks, the animals were sacrificed and the scaffolds were taken out for tissue sectioning, and the cellular growth was observed by HE and Masson staining.
In physical assay, both the diameter and size of nanofiber holes were increased in the PCL-SF scaffold when the proportion of SF is increased. There is no difference for degradation rate under cell culture medium soaking for 8 weeks which was 10% less in dry weight. The tensile strength of PCL-SF nanofibers increased when the PCL ratio increased. Typical spectrum peaks for PCL and SF were observed in the spectra of PCL/SF blends. MTT result indicates that the incorporation of SF into PCL was beneficial for cell proliferation and parallel to the percentage of SF ratio and 4:6 of PCL-SF scaffold is the best ratio for cellular growth. Higher cellular proliferation was seen in 4:6 of 3D-ES PCL-SF scaffolds than 4:6 of ES PCL-SF scaffolds due to increased diameter of nanofibers and size of holes. The PCL-SF scaffold anastomosis in ex vivo bath showed cellular growth stably along the scaffold for two weeks and most of the cells grow along the outboard. In animal model, after 6 and 7 weeks, different cells can be observed to grow along the outboard of the scaffold, from the lumen outward: Mucosa, Submucosa, muscular layer and the serosa layer, mucosal layer growth in the scaffold inner edge of proximal (kidney) side, Mucosa and muscular layer growth in the scaffold inner edge of distal (bladder) side, However, in the middle of the inner edge of the scaffold, the cells had not grown yet till 7 weeks.
In our study, 3D-ES produced 4:6 PCL-SF nanofiber scaffolds are suitable for cell tissue growth, and achieve the purpose of ureteral reconstruction in animal experiments. Therefore this new form scaffold can be used as clinical urinary tract tissue reconstruction in the future.

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